Epidemic typhus, one of the most serious bacterial diseases affecting humans, is caused by the louseborne pathogen, Rickettsia prowazekii. Due to the high mortality rate of this disease, the lack of an effective vaccine, and the possibility o aerosol dissemination, R. prowazekii is designated as a select agent and has the potential to pose a severe threat to public health and safety. R. prowazekii is an obligate intracellular, parasitic bacterium that grows only within the cytosol of the host cell, unbounded by a vacuolar membrane. This ability to exploit the intracellular environment and cause human disease provides the basis for our studies designed to elucidate the mechanisms underlying obligate intracellular growth and R. prowazekii virulence. This proposal addresses the identification of critical stage-specific genes that are regulated as a rickettsial infection proceeds from a few rickettsiae per cell to hundreds per cell. A major hurdle in this field is the heterogeneity in the bacterial load within an infected population and the inability to generate synchronous rickettsial cultures. To tackle this problem, we propose to use a fluorescence activated cell sorting (FACS) approach to separate R. prowazekii-infected host cells, based on bacterial load, into distinct stage-specific populations. Using genetically-modified GFP-expressing R. prowazekii, we have demonstrated that FACS can be used to separate R. prowazekii-infected host cells into distinct populations harboring uniform numbers of rickettsiae per cell (population gating). We hypothesize that FACS-based separation of rickettsiae-infected cells will facilitate identification of rickettsial genes that are regulated at specific growth stages and that play key roles in optimizing the growth, virulence, and subsequent spread of this intracellular pathogen. We will address this hypothesis through the following specific aims: In Aim 1 we will identify and separate R. prowazekii-infected host cells into distinct populations according to bacterial load. Rickettsial numbers in isolated populations will be evaluated by microscopy and quantitative PCR. Optimization will include the evaluation of a codon-adapted fluorescent protein. We will evaluate different host cell lines (e.g. vertebrate and arthropod) and a virulent rickettsial strain. The effect of initial host cell infection levels and harvest times wll be examined. In Aim 2 we will identify, at both the transcriptional (gene arrays) and protein level (differential proteomics), genes that are differentially expressed as the rickettsiae transition frm exponential growth to lysis stage. Completion of this project will significantly advance the field, providing an accurate assessment of gene expression at defined stages in the R. prowazekii intracellular growth cycle. This data will lay a foundation for further delineation of molecular mechanisms underlying the infection, intracellular growth, and virulence of R. prowazekii and may reveal new targets for design of novel therapeutics to combat this resourceful and dangerous pathogen.